Metabolically engineered bacterial strains having non-functional endogenous gluconate transporters

ABSTRACT

The present invention relates to engineering metabolic pathways in bacterial host cells which results in enhanced carbon flow for the production of ascorbic acid (ASA) intermediates. In particular, the invention relates to increasing the production of ASA intermediates in bacterial cells by enhancing the availability of gluconate resulting from the inactivation of endogenous gluconate transporter genes.

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 60/473,310, filed May 22, 2003 and to U.S.Provisional Patent Application Ser. No. 60/478,056, filed Jun. 11, 2003.

FIELD OF THE INVENTION

The present invention relates to engineering metabolic pathways inbacterial host cells which results in enhanced carbon flow for theproduction of polyols and keto-derivatives thereof. More specificallythe invention relates to the enhanced production of keto-polyols, suchas sugar-keto acids and more specifically to ascorbic acidintermediates. In particular, the invention relates to enhancing theindustrial production of gluconate, 2-keto-D-gluconic acid (KDG or2-KDG), 2,5-diketogluconate (DKG or 2-DKG) and 2-keto-L-gulonic acid(KLG or 2-KLG) in Pantoea cells by altering an endogenous gluconatetransporter. The invention further relates to altered Pantoea strainshaving one or more non-functional endogenous gluconate transporter genesand optionally inactivated endogenous glucokinase and/or gluconokinasegenes.

BACKGROUND OF THE INVENTION

Numerous products of commercial interest, such as intermediates ofL-ascorbic acid, have been produced biocatalytically in geneticallyengineered host cells. L-Ascorbic acid (vitamin C, ASA) is commonly usedin the pharmaceutical and food industries as a vitamin and antioxidant,and due to this relatively large market volume and high value as aspecialty chemical the synthesis of ASA has received considerableattention.

A chemical synthesis route from glucose to ASA, commonly known as theReichstein-Grussner method, was first disclosed in 1934 (Reichstein T.et al., (1934) Helv. Chim. Acta., 17:311-328 and Reichstein T. et al.(1933) Helv. Chim. Acta. 16: 561, 1019). A bioconversion method for theproduction of an ASA intermediate, 2-KLG, has been disclosed by Lazaruset al. (1989, “Vitamin C: Bioconversion via a Recombinant DNA Approach”,GENETICS AND MOLECULAR BIOLOGY OF INDUSTRIAL MICROORGANISMS, AmericanSociety for Microbiology, Washington D.C. Edited by C. L. Hershberger).This bioconversion of a carbon source to KLG involves a variety ofintermediates, and the enzymatic process is associated with co-factordependent 2,5-DKG reductase activity (DKGR). Additionally, recombinantDNA techniques have been used to bioconvert glucose to KLG in Erwiniaherbicola in a single fermentative step (Anderson, S. et al., (1985)Science 230:144-149). Effective procedures for converting KLG to ASA aredescribed in Crawford et al., ADVANCES IN CARBOHYDRATE CHEMISTRY ANDBIOCHEMISTRY, 37:79-155 (1980).

A number of strategies have been explored in bacteria and othermicroorganisms to increase the production of ASA intermediates. Some ofthese strategies include; gene deletions, gene additions and randommutagenesis. In particular, some gene manipulation strategies arementioned below and include: (i) overexpressing particular genes in theASA pathway (Anderson et al., (1985) Sci. 230:144-149; Grindley et al.,(1988) Appl. Environ. Microbiol. 54: 1770; and U.S. Pat. No. 5,376,544);(ii) mutating genes encoding glycolytic enzymes (Harrod, et al. (1997)J. Ind. Microbiol. Biotechnol. 18:379-383; Wedlock, et al. (1989) J.Gen. Microbiol. 135: 2013-2018; and Walsh et al. (1983) J. Bacteriol.154:1002-1004); (iii) utilizing bacterial host strains deficient inglucokinase (Japanese patent publication JP 4267860; Russell et al.(1989) Appl. Environ. Microbiol. 55: 294-297; Barredo et al. (1988)Antimicrob. Agents-Chemother 32: 1061-1067; and DiMarco et al. (1985)Appl. Environ. Microbiol. 49:151-157); (iv) reducing metabolism ofglucose or gluconate by deletion of a gene required for phosphorylation,for example deleting the glucokinase gene (glkA) or gluconokinase gene(gntK) (WO 02/081440); and (v) reducing metabolism by manipulatingenzymes involved in carbon utilization downstream of the initial glucosephosphorylation, for example manipulating phosphoglucose isomerase,phosphofructokinase, glucose-6-phosphate dehydrogenase,6-phosphogluconate dehydrogenase or 6-phosphogluconate dehydratase.

Despite the above strategies, problems still persist concerning thediversion of ASA intermediates for catabolic metabolic purposes, andthis results in reducing the efficiency and overall production of ASAintermediates. Thus, there still remains a need for improved productionmethods for ASA intermediates which are coupled to the metabolicpathways of host cells.

Carbon sources, such as glucose and gluconate, involved in theproduction of ASA intermediates may be separated by a cellular membranefrom the reactions which utilize these substrates. When such a substrateand the synthetic machinery are separated, production of the product mayrequire translocation of the substrate to the site of the syntheticreaction. Alternatively products generated inside the cell may requiretranslocation from within the cell. Therefore, altering a substratetransport system may result in increased or decreased substrateavailability for a particular metabolic pathway.

The present invention provides altered bacterial strains which includenon-functional gluconate transporter molecules. The altered bacterialstrains, which include the non-functional transporter molecules, have anincreased amount of carbon substrate, such as glucose that may beutilized for the production of desired products, such as ASAintermediates.

SUMMARY OF THE INVENTION

The present invention provides methods of producing a desired productwhich comprises manipulating bacterial host cells to reduce carbonsubstrate diverted to metabolic pathways, thus increasing theproductivity of the host cell for a desired product. Such manipulatedbacterial host cells when cultured in the presence of a carbon sourcedemonstrate increased yield of a desired product as measured directlyand/or indirectly.

In one aspect, the invention provides isolated polynucleotides encodingpolypeptides isolated from a strain of Pantoea having gluconatetransporter activity. In one embodiment, the isolated polynucleotidecomprises a nucleotide sequence having at least 40% sequence identity toa nucleotide sequence selected from the group consisting of SEQ ID NO:1and SEQ ID NO:3. In a second embodiment, the isolated polynucleotidecomprises a nucleotide sequence having at least 80% sequence identity toa nucleotide sequence selected from the group consisting of SEQ ID NO:1and SEQ ID NO:3. In a third embodiment, the isolated polynucleotidecomprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. In afourth embodiment, the isolated polynucleotide comprises a nucleotidesequence which encodes a gluconate transporter having an amino acidsequence at least 40% identical to the sequence of SEQ ID NO:2. In afifth embodiment, the isolated polynucleotide comprises a nucleotidesequence which encodes a gluconate transporter having an amino acidsequence at least 80% identical to the sequence of SEQ ID NO:2. In asixth embodiment, the isolated polynucleotide comprises a nucleotidesequence which encodes a gluconate transporter having an amino acidsequence at least 80% identical to the sequence of SEQ ID NO:4. In aseventh embodiment, the isolated polynucleotide comprises a nucleotidesequence which encodes a gluconate transporter having an amino acidsequence at least 90% identical to the sequence of SEQ ID NO:4. In aneighth embodiment the isolated polynucleotide encodes a gluconatetransporter having the sequence of SEQ ID NO:2 or SEQ ID NO:4.

In another aspect, the invention relates to an isolated gluconatetransporter protein comprising an amino acid sequence shown in SEQ IDNO:2 or an amino acid sequence having at least 80% amino acid sequenceidentity thereto. In one embodiment, the isolated gluconate transporterprotein has been isolated from Pantoea. In a second embodiment, theisolated gluconate transporter protein has the sequence shown in SEQ IDNO:2.

In a further aspect, the invention relates to an isolated gluconatetransporter protein comprising an amino acid sequence shown in SEQ IDNO:4 or an amino acid sequence having at least 80% amino acid sequenceidentity thereto. In one embodiment, the isolated gluconate transporterprotein has been isolated from Pantoea. In a second embodiment, theisolated gluconate transporter protein has the sequence shown in SEQ IDNO:4.

In another aspect, the present invention provides a method for enhancingthe level of production of an ascorbic acid (ASA) intermediatecomprising, obtaining an altered bacterial host cell which is capable ofproducing an ASA intermediate, wherein an endogenous gluconatetransporter gene has been rendered non-functional, culturing the alteredbacterial host cell under suitable culture conditions in the presence ofa carbon source to allow the production of an ASA intermediate andobtaining the ASA intermediate, wherein the level of production of theASA intermediate is enhanced compared to the level of production of theASA intermediate in a corresponding unaltered bacterial cell grown underessentially the same conditions. In one embodiment of the process, theendogenous gluconate transporter has at least 40% sequence identity tothe amino acid sequence of SEQ ID NO:2 or at least 80% sequence identityto the amino acid sequence of SEQ ID NO:4. In a second embodiment of theprocess, the bacterial host cell is altered to include twonon-functional gluconate transporter genes. In a third embodiment of theprocess, the gluconate transporter gene is deleted from the hostchromosome. In a fourth embodiment, the process further comprises thestep of isolating the ASA intermediate. In a fifth embodiment, theprocess further comprises converting the ASA into a second product. In asixth embodiment, the host cell is an Enterobacteriaceae cell. In aseventh embodiment of the process, the endogenous gluconate transportergene encodes a protein having at least 80% sequence identity to thesequence of SEQ ID NO:2 or SEQ ID NO:4. In an eighth embodiment of theprocess, the gluconate transporter gene encodes a protein designatedGntU having the amino acid sequence of SEQ ID NO:2. In an ninthembodiment of the process, the gluconate transporter gene encodes aprotein designated IdnT having an amino acid sequence of SEQ ID NO:4. Ina tenth embodiment of the process, the bacterial host cell includes aninactivated glucokinase (glk) gene and/or an inactivated gluconokinase(gntK) gene. In an eleventh embodiment, the invention concerns analtered bacterial host cell produced by said process.

In a further aspect, the invention provides a recombinantEnterobacteriaceae strain comprising one or more nonfunctionalendogenous gluconate transporter proteins. In one embodiment, the one ormore non-functional endogenous gluconate transporter proteins has anamino acid sequence of at least 80% sequence identity to the sequence ofSEQ ID NO:2 or SEQ ID NO:4. In further embodiments of this aspect therecombinant Enterobacteriaceae strain further comprises an inactivatedendogenous glucokinase gene, an inactivated endogenous gluconokinasegene, and/or an overexpressed or heterologous DKG transporter genes. Ina preferred embodiment, the recombinant strain is a Pantoea or E. colistrain.

In yet another aspect, the invention provides a recombinant Pantoea cellcomprising an endogenous gluconate transporter gene which has beeninactivated, wherein said gluconate transporter gene prior to beinginactivated encoded a gluconate transporter protein comprising the aminoacid sequence shown in SEQ ID NO:2 or an amino acid sequence having atleast 40% sequence identity thereto, wherein said protein functioned bytransporting gluconate across a Pantoea cell membrane into thecytoplasm. In one embodiment, the recombinant Pantoea cell will includean inactivated endogenous glucokinase gene and in another embodiment therecombinant Pantoea cell will include a heterologous DKG transportergene.

In another aspect, the invention provides an altered Pantoea cellcomprising an inactivated endogenous gluconate transporter gene, whereinsaid gluconate transporter gene prior to being inactivated encoded agluconate transporter protein comprising the amino acid sequence shownin SEQ ID NO:4 or an amino acid sequence having at least 80% sequenceidentity thereto, wherein said protein functioned by transportinggluconate across a Pantoea cell membrane into the cytoplasm. In oneembodiment, the altered Pantoea cell will include an inactivatedglucokinase gene and in another embodiment the altered Pantoea cell willinclude a modified DKG transporter gene.

In a further aspect, the invention provides a method of enhancing theproduction of an ascorbic acid (ASA) intermediate from a carbon sourcecomprising, obtaining a Pantoea host cell comprising an endogenousgluconate transporter gene which encodes a gluconate transporter proteinhaving an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or anamino acid sequence having at least 40% sequence identity to SEQ ID NO:2or SEQ ID NO:4, altering the Pantoea host cell by rendering thegluconate transporter gene non-functional, wherein the altered Pantoeahost cell is capable of producing an ASA intermediate in the presence ofglucose, culturing said altered Pantoea host cell under suitableconditions in the presence of a carbon source, and allowing theproduction of an ASA intermediate from the carbon source, wherein theproduction of said ASA intermediate is enhanced in the altered Pantoeacell compared to the production of the ASA intermediate in an unalteredPantoea host cell cultured under essentially the same cultureconditions. In one embodiment of the method, the ASA intermediate isselected from the group consisting of gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, 2-keto-L-gulonic acid and L-idonic acid. In asecond embodiment, the ASA intermediate is further converted toerythorbic acid or tartrate. In a third embodiment, the gluconatetransporter gene is gntU having the nucleic acid sequence illustrated bySEQ ID NO:1 and/or idnT having the nucleic acid sequence illustrated inSEQ ID NO:3.

In still another aspect, the invention provides a method of increasingthe production of KLG in a Pantoea host cell which comprises obtaining aPantoea host cell including a gluconate transporter gene which encodes agluconate transporter having an amino acid sequence shown in SEQ IDNO:2, SEQ ID NO:4 or an amino acid sequence having at least 40% sequenceidentity to SEQ ID NO:2 or SEQ ID NO:4; altering the Pantoea host cellby rendering the gluconate transporter gene non-functional, wherein thealtered Pantoea host cell is capable of producing KLG in the presence ofglucose; culturing said altered Pantoea host cells under suitableconditions in the presence of a carbon source; and allowing theproduction of KLG from the carbon source, wherein the production of saidKLG is enhanced in the altered Pantoea host cell compared to theproduction of KLG in an unaltered Pantoea host cell cultured underessentially the same culture conditions. In one embodiment, theinvention relates to the altered Pantoea cell obtained according to theabove method. In further embodiments, the method comprises a Pantoeacell genetically engineered to include a non-functional glucokinasegene, a non-functional gluconokinase gene and/or overexpressed DKGtransporter genes.

In still another aspect, the invention provides a method of increasingthe production or availability of gluconate in an oxidative pathway of aPantoea host cell which comprises obtaining a Pantoea host cellincluding a gluconate transporter gene which encodes a gluconatetransporter having an amino acid sequence shown in SEQ ID NO:2, SEQ IDNO:4 or an amino acid sequence having at least 40% sequence identity toSEQ ID NO:2 or SEQ ID NO:4; altering the Pantoea host cell by renderingthe gluconate transporter gene non-functional, wherein the alteredPantoea host cell is capable of producing gluconate in the presence ofglucose; culturing said altered Pantoea host cells under suitableconditions in the presence of a carbon source; and allowing theproduction of gluconate from the carbon source, wherein the productionor availability of said gluconate in an oxidative pathway is enhanced inthe altered Pantoea host cell compared to the production or availabilityof gluconate in an unaltered Pantoea host cell cultured underessentially the same culture conditions. In one embodiment, theinvention relates to the altered Pantoea cell obtained according to theabove method. In further embodiments, the method comprises a Pantoeacell genetically engineered to include a non-functional glucokinasegene, a non-functional gluconokinase gene and/or overexpressed DKGtransport genes.

Another aspect of the invention provides a method of increasing theproduction of KDG in a Pantoea host cell which comprises obtaining aPantoea host cell including a gluconate transporter gene which encodes agluconate transporter having an amino acid sequence shown in SEQ IDNO:2, SEQ ID NO:4 or an amino acid sequence having at least 40% sequenceidentity to SEQ ID NO:2 or SEQ ID NO:4; altering the Pantoea host cellby rendering the gluconate transporter gene non-functional, wherein thealtered Pantoea host cell is capable of producing KDG in the presence ofglucose; culturing said altered Pantoea host cells under suitableconditions in the presence of a carbon source; and allowing theproduction of KDG from the carbon source, wherein the production of saidKDLG is enhanced in the altered Pantoea host cell compared to theproduction of KDG in an unaltered Pantoea host cell cultured underessentially the same culture conditions. In one embodiment the inventionrelates to the altered Pantoea cell obtained according to the abovemethod. In further embodiments, the method comprises a Pantoea cellgenetically engineered to include a non-functional glucokinase gene, anon-functional gluconokinase gene and/or overexpressed DKG transportgenes. In some embodiments, the invention includes the altered Pantoeahost cells.

In still another aspect, the invention provides a method of increasingthe production of DKG in a Pantoea host cell which comprises obtaining aPantoea host cell including a gluconate transporter gene which encodes agluconate transporter having an amino acid sequence shown in SEQ IDNO:2, SEQ ID NO:4 or an amino acid sequence having at least 40% sequenceidentity to SEQ ID NO:2 or SEQ ID NO:4; altering the Pantoea host cellby rendering the gluconate transporter gene non-functional, wherein thealtered Pantoea host cell is capable of producing DKG in the presence ofglucose; culturing said altered Pantoea host cells under suitableconditions in the presence of a carbon source; and allowing theproduction of DKG from the carbon source, wherein the production of saidDKG is enhanced in the altered Pantoea host cell compared to theproduction of DKG in an unaltered Pantoea host cell cultured underessentially the same culture conditions. In one embodiment the inventionrelates to the altered Pantoea cell obtained according to the abovemethod. In further embodiments, the method comprises a Pantoea cellgenetically engineered to include a non-functional glucokinase geneand/or a non-functional gluconokinase gene. In yet another aspect, theinvention provides a method for increasing the availability of gluconatefor oxidative pathway production of an ascorbic acid (ASA) intermediatewhich comprises obtaining an altered bacterial host cell, wherein anendogenous gluconate transporter gene has been rendered non-functional;culturing the altered bacterial host cell under suitable cultureconditions in the presence of a carbon source to allow the production ofan ASA intermediate; and obtaining the ASA intermediate, wherein the ASAintermediate is selected from the group consisting of 2-keto-D-gluconate(2-KDG), 2,5-diketo-D-gluconate (2,5-DKG), 2-keto-L-gulonic acid(2-KLG), 5-keto-D-gluconate (5-KDG) and L-idonic acid (IA) and theendogenous gluconate transporter gene encodes a gluconate transporterhaving the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or a sequencehaving at least 80% identity to either SEQ ID NO:2 or SEQ ID NO:4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the oxidative pathway for the production of ascorbic acid(ASA). E1 stands for glucose dehydrogenase (GDH); E2 stands for gluconicacid dehydrogenase (GAD H); E3 stands for 2-keto-D-gluconic aciddehydrogenase (KDGH); and E4 stands for 2,5-diketo-D-gluconic acidreductase (2,5 DKGR).

FIG. 2 provides a schematic representation of some of the metabolicroutes involved in glucose assimilation in a bacterial host cell such asPantoea citrea. The following abbreviations have been used in the figureand are applied throughout the disclosure: glucose dehydrogenase=(GDH);gluconic acid dehydrogenase=(GADH); 2-keto-D-gluconate=(2-KDG or KDG);2-keto-D-gluconic acid dehydrogenase=(2-KDGH or KDGH);2,5-diketogluconate=(2,5-DKG or DKG); 2,5-diketo-D-gluconic acidreductase=(2, 5 DKGR); 2-keto-L-gulonic acid=(2-KLG or KLG);2-ketoreductase=(2KR or KR); 5-ketoreductase=(5KR or KR);5-keto-D-gluconate=(5-KDG); idonate dehydrogenase ═(IADH);glucokinase=(GlkA or Glk) and gluconokinase=(GntK). Boxes labeled with a“T” represent putative transporters and boxes labeled with an “E”represent putative efflux systems. Boxes labeled with a “1” or “2”represent putative gluconate transporters according to the invention.

As observed in FIG. 2, in a microbial cell there are multipleconnections between oxidative and catabolic pathways, such as theglycolytic pathway, the pentose pathway, the tricarboxylic acid (TCA)cycle pathway and ASA intermediate production pathway. As can be seen,the inactivation of a gluconate transporter (1) and (2) blocks entry ofgluconate into the cytoplasm resulting in a decrease in the amount ofgluconate available for phosphorylation by GntK in the pentoseassimilation pathway and resulting in a decrease in the amount ofgluconate available for enzymatic reduction by 5-KR or 2-KR in thecytoplasm. Further it can be seen that inactivation of a gluconatetransporter (1) also blocks entry of idonate into the cytoplasm andhence results in more substrate being made available for production ofASA intermediates such as KLG.

FIG. 3 depicts a nucleic acid sequence (SEQ ID NO:1) for a Pantoeacitrea gluconate transporter gene (gntU).

FIG. 4 depicts an amino acid sequence (SEQ ID NO:2) for a Pantoea citreagluconate transporter protein (GntU).

FIG. 5 depicts a nucleic acid sequence (SEQ ID NO:3) for a Pantoeacitrea gluconate transport gene (idnT).

FIG. 6 depicts an amino acid sequence (SEQ ID NO:4) for a Pantoea citreagluconate transporter protein (IdnT).

FIG. 7 is a general schematic illustrating the process and vectors usedto inactivate a chromosomal gluconate transporter gene in a strain ofPantoea citrea by homologous recombination using a non-replicating R6Kvector, wherein gntU is the gene encoding a gluconate transporterprotein, gntK is the gene coding for glucokinase, NsiI is a restrictionsite, loxP is a recombinase recognition sequence and Cm^(R) is the genefor the selective marker chloramphenicol. Reference is also made toexample 1.

FIG. 8 is a general schematic illustrating the process and vectors usedto inactivate a gluconate transporter chromosomal gene in a strain ofPantoea citrea by homologous recombination using a non-replicating R6Kvector, wherein idnD is the gene coding for idonate dehydrogenase, idnTis the gene encoding a gluconate transporter protein, NsiI and BgII arerestriction sites, loxP is a recombinase recognition sequence and Cm^(R)is the gene for the selective marker chloramphenicol. The plasmid,pEkIdnDTcat2 was used for the transformation of the Pantoea host cell.Reference is also made to example 2.

FIG. 9 depicts the results from gluconate uptake rate experiments(nmole/OD/sec) for three different P. citrea strains. Strain 139-2A/pD92was used as a control and was derived from ATCC Accession No. 39140.Strain MDP41/pD92-A was derived from 139-2A/pD92 and includes aninterruption of glkA. Strain MDP41UDT/pD92-A was derived fromMDP41/pD92-A (according to the teachings of examples 1 and 2) andincludes, in addition to an interruption of glkA, a gntN deletion and anidnT deletion.

FIG. 10 depicts the percent sequence identity between 10 gluconatetransporters. The amino acid sequence as set forth in SEQ ID NO:2 isdesignated Pc-gntU. The percent identity between Pc-gntU and the othergluconate transporters is 34% or less. The amino acid sequence as setforth in SEQ ID NO:4 is designated Pc-idnT. The percent identity betweenPc-idnT and the other gluconate transporters is 76.3% or less. Thepercent amino acid sequence identity between SEQ ID NO:2 and SEQ ID NO:4is 33%. Gluconate transports Ec-yjhF, Ec-dsdX, Ec-gntP, Ec-gntT,Ec-gntU, Ec-idnT and Ec-0454 are obtained from E. coli and Bs-gntP isobtained from Bacillus.

FIG. 11 generally depicts some of the metabolic pathways involved in thesynthesis of various polyols that are of commercial relevance.

DETAILED DESCRIPTION OF THE INVENTION

Recombinant bacterial host cells have been constructed which producepolyols, particularly keto-polyols and more particularly ASAintermediates, and which are altered to reduce carbon substrate divertedto catabolic pathways. Possible catabolic routes which can be used todivert glucose into cellular metabolism involving at least one enzymaticstep, include but are not limited to the formation of each of thefollowing: glucose-6-P, fructose, sorbitol, gluconate-6-P, idonate,sorbosone, deoxy-gluconate, 2,5-di-keto-gluconate, and2-keto-D-gluconate, 5-keto-D-gluconate and 2-keto-3-deoxygluconate. (SeeFIGS. 1 and 2).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, and biochemistry,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, MOLECULAR CLONING: A LABORATORYMANUAL, second edition (Sambrook et al., 1989) Cold Spring HarborLaboratory Press; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubelet al., eds., 1987 and annual updates); OLIGONUCLEOTIDE SYNTHESIS (M. J.Gait, ed., 1984); PCR: THE POLYMERASE CHAIN REACTION, (Mullis et al.,eds., 1994); and MANUAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY,Second Edition (A. L. Demain, et al., eds. 1999).

DEFINITIONS

Unless defined otherwise herein, all technical terms and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Singleton,et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., JohnWiley and Sons, New York (1994) and Hale and Marham, THE HARPERDICTIONARY OF BIOLOGY, Harper Perennial, New York (1991) provide one ofskill with general dictionaries of many of the terms used in thisinvention.

As used herein the term “polyol” means an alcohol molecule with numeroushydroxyl groups. A “keto-poly derivative” is a polyol which includes oneor more keto groups in the polyol molecule. Examples of polyols includebut are not limited to glucose, fructose, mannose, idose, galactose,sorbose, ribose, altose, arabinose, erythrose, xylose, xylulose,maltose, sucrose, erythrulose, gluconate, idonate, gulonate,galacturonate, mannitol, sorbitol, sorbosone, glycerol, erythritol,arabitol, DKG, KDG, KLG, gluconolactone and maltitol. The compounds mayoccur in either the D or L configuration.

As used herein an “ascorbic acid (ASA) intermediate” means a biochemicalcapable of being converted to ASA by enzymatic or chemical means andincludes, but is not limited to, gluconate (GA); 2-keto-D-gluconate(2-KDG or KDG); 2,5-diketo-D-gluconate (2,5-DKG or DKG);2-keto-L-gulonic acid (2KLG or KLG); 5-keto-D-gluconate (5-KDG) andL-idonic acid (IA).

As used herein, the term “carbon source” encompasses-suitable carbonsubstrates ordinarily used by microorganisms, such as 6 carbon sugars,including but not limited to glucose (G), gulose, sorbose, fructose,idose, galactose and mannose all in either D or L form, or a combinationof 6 carbon sugars, such as glucose and fructose, and/or 6 carbon sugaracids including but not limited to 2-keto-L-gulonic acid, idonic acid(IA), gluconic acid (GA), 6-phosphogluconate, 2-keto-D-gluconic acid (2KDG), 5-keto-D-gluconic acid, 2-ketogluconatephosphate,2,5-diketo-L-gulonic acid, 2,3-L-diketogulonic acid, dehydroascorbicacid, erythorbic acid (EA) and D-mannonic acid or the enzymaticderivatives thereof.

As used herein, the phrase “transport system” refers to at least onemacromolecule, such as a protein, which is located in a cell membraneand is involved in the translocation of a chemical compound, such as acarbon substrate, across a cell membrane and into or out from a cell orcellular compartment. A transport system may include two, three, four,five, six or more macromolecules, such as proteins. These proteins mayor may not be simultaneously induced by a substrate. (Reference is madeto Saier M. et al., (1998) in ADVANCES IN MICROBIAL PHYSIOLOGY (Poole R.K. ed) pages 81-136, Academic Press, San Diego, Calif. for a discussionof the classification of transporters). In the literature, a transportersystem is sometimes referred to as a permease. For example, thegluconate permease (GntP) family in E. coli is encoded by seventransporter genes which include gntP, gntT, gntU, gntW, ORF f449, dsdxand ORFo454 (Peekhaus et al., (1997) FEMS Micro. Lett. 147:233-238).Reference is also made to Yamada et al., (1996) Biosci. Biotech.Biochem., 60:1548-1550 and Tsunedomi et al., (2003) J. Bacteriol.185:1783-1795. In one embodiment, a transporter protein as used hereinis a “symporter”. A symporter is defined as a transporter thattransports two or more substrates or species together in the samedirection in a coupled process using an electrochemical potentialgradient. One example of a symporter is a proton coupled substratetransporter.

The term “gluconate transporter” as used herein refers to a protein thatcatalyzes the transport of gluconate into the cytoplasm across theinternal cell membrane. In addition to gluconate, a gluconatetransporter may catalyze the transport of other sugar acid molecules orsugar-keto acids across a cell membrane.

A “sugar acid molecule” is defined as a sugar molecule having one ormore acid groups. Preferred sugar acids that may be transported by agluconate transporter according to the invention will not only includegluconate, but may also include idonic acid, galacturonic acid,glucuronic acid and/or gulonic acid.

A “sugar-keto acid” is defined as a sugar molecule having one or moreketo groups and one or more acid groups. A gluconate transporteraccording to the invention may also catalyze the transport of sugar ketoacids, such as 2-keto-D-gluconic acid (2-KDG or KDG), 2-keto-L-gulonicacid (KLG), 5-keto-D-gluconic acid (5-KDG), D-tagaturonic acid and/orD-fructuroinc acid.

The term “KDG transporter” as used herein refers to a protein thatcatalyzes the transport of KDG across a cell membrane. More specificallythe KDG transporter facilitates the entry of KDG into the cytoplasmacross the internal cell membrane.

The term “glucose transporter” as used herein refers to a protein thatcatalyzes the transport of glucose across a cell membrane. Morespecifically the glucose transporter facilitates the entry of glucoseinto the cytoplasm across the internal cell membrane.

The term “DKG transporter” as used herein refers to a protein thatcatalyzes the transport of DKG across a cell membrane. More specificallythe DKG transporter facilitates the entry of DKG into the cytoplasmacross the internal cell membrane.

“Cytoplasm” or “cytoplasmic” refers to being within the inner cellmembrane. Extracellular or outside the inner cell membrane refers tocell locations on the opposite side of a membrane from the cytoplasm,including but not limited to the periplasm. Internal or inner membrane(and sometimes referred to as the periplasmic membrane) refers to thebarrier that separates the cytoplasm from the periplasm. Intracellularrefers to the portion of the cell on the side of the membrane that isclosest to the cytosol. Intracellular includes cystolic.

As used herein the term “gene” means a DNA segment that is involved inproducing a polypeptide and includes regions preceding and following thecoding regions as well as intervening sequences (introns) betweenindividual coding segments (exons). As used herein when describingproteins and genes that encode them, the term for the gene is notcapitalized and is italics, i.e. g/kA. The term for the protein isgenerally not italicized and the first letter is capitalized, i.e. GlkA.

The terms “protein” and “polypeptide” are used interchangeabilityherein.

As used herein “oxidative pathway” of a host cell means that a host cellcomprises at least one enzyme that oxidizes a carbon source, such asD-glucose and/or its metabolites. An oxidative pathway in a host cellmay comprise, one, two, three or more enzymes. An example of anoxidative pathway is the formation of gluconate from glucose through theactivity of glucose dehydrogenase. Another example of an oxidativepathway is the formation of DKG from glucose through the activity ofglucose dehydrogenase, gluconate dehydrogenase and 2-KDG dehydrogenase.

As used herein “catabolic pathway” of a host cell means that a host cellcomprises at least one enzyme that generates at least one metabolicintermediate. Very often, the formation of the metabolic intermediate iscoupled to the generation of ATP, NADPH, or NADH for example byphosphorylating a carbon source such as D-glucose and/or itsmetabolites. An intracellular catabolic pathway in a host cell means thehost cell comprises the activity of at least one enzyme in the host cellcytosol. In some embodiments, a catabolic pathway comprises the activityof two, three or more enzymes. Catabolic pathways include but are notlimited to glycolysis, the pentose pathway and the TCA cycle pathway(FIGS. 1 and 2).

As used herein, the phrase “glucokinase” (E.C.-2.7.1.2) means an enzymewhich phosphorylates D-glucose or L-glucose at its 6th carbon. As usedherein, the phrase “gluconokinase” (E.C.-2.7.1.12) means an enzyme whichphosphorylates D-gluconate or L-gluconate at its 6^(th) carbon.

The term “nonfunctional”, “inactivated” or “inactivation” when referringto a gene or a protein means that the known normal function or activityof the gene or protein has been eliminated or highly diminished. Forexample, inactivation of a gluconate transporter can be effected byinactivating the gntU and/or idnT chromosomal genes. Inactivation whichrenders the gene or protein non-functional includes such methods asdeletions, mutations, substitutions, interruptions or insertions in thenucleic acid gene sequence.

A “deletion” of a gene as used herein may include deletion of the entirecoding sequence, deletion of part of the coding sequence, deletion ofthe regulatory region, deletion of the translational signals or deletionof the coding sequence including flanking regions.

As used herein the term “mutation” when referring to a nucleic acidrefers to any alteration in a nucleic acid such that the product of thatnucleic acid is partially or totally inactivated. Examples of mutationsinclude but are not limited to point mutations, frame shift mutationsand deletions of part or all of a gene encoding a transporter.

As used herein, the term “modified” when referring to nucleic acid or apolynucleotide means that the nucleic acid has been altered in some wayas compared to a wild type nucleic acid, such as by mutation in;deletion of part or all of the nucleic acid; or by being operably linkedto a transcriptional control region.

An “altered bacterial host cell or strain” according to the invention isa bacterial cell having an inactivated gluconate transporter. In oneembodiment, an altered bacterial host cell will have an enhanced levelof expression (production) over the level of production of the samedesired product in a corresponding unaltered bacterial host grown underessentially the same growth conditions. In other embodiments, an alteredhost cell will have an enhanced level of availability of gluconate to beused by the host cell to produce ASA intermediates as compared to acorresponding unaltered bacterial host cell.

An “unaltered bacterial host cell or strain” is a bacterial cell, whichgenetically corresponds to an altered bacterial host cell or strain butwherein the endogenous gene encoding the gluconate transporterencompassed by the invention is not inactivated and remains functional.

The “enhanced level of production” refers to an increased yield of thedesired end-product, or amount of substrate as compared to the yieldfrom an unaltered bacterial host when cultured under essentially thesame conditions. For example an increase of 2%, 5%, 10%, 15%, 20%, 30%,40% or more over the yield of the unaltered bacterial host. Yield, maybe expressed in numerous ways including as a weight % (gm product/gmsubstrate) or gm/L per unit of time. The enhanced level of productionresults from the inactivation of one or more chromosomal genes encodinga gluconate transporter according to the invention. In a firstembodiment, the enhanced level of production results from the deletionof one or more chromosomal genes encoding a gluconate transporter. In asecond embodiment, the enhanced level of production results from theinsertional inactivation (interruption) of one or more chromosomal genesencoding a gluconate transporter.

As used herein “chromosomal integration” is a process whereby anintroduced polynucleotide is incorporated into a host cell chromosome.The process preferably takes place by homologous recombination.

As used herein, “modifying” the level of protein or enzyme activityproduced by a host cell refers to controlling the levels of protein orenzymatic activity produced during culturing, such that the levels areincreased or decreased as desired.

“Under transcriptional control” or “transcriptionally controlled” areterms well understood in the art that indicate that transcription of apolynucleotide sequence, usually a DNA sequence, depends on its beingoperably linked to an element which contributes to the initiation of, orpromotes, transcription.

“Operably linked” refers to a juxtaposition wherein the elements are inan arrangement allowing them to function. “Under translational control”is a term well understood in the art that indicates a regulatory processthat occurs after the messenger RNA has been formed. “Expression”includes transcription and/or translation. The term “overexpressed”means an increased number of copies of the same gene in a host cellgenome.

“Allowing the production of an ASA intermediate from a carbon source,wherein the production of said ASA intermediate is enhanced compared tothe production of the ASA intermediate in a corresponding unalteredbacterial host cell or strain” means contacting the carbon source, withan altered bacterial strain to produce the desired end-product. Byaltering a gluconate transporter according to the invention, an alteredbacterial host can demonstrate enhanced desired end-product production.

The phrase “desired end-product” or “desired product” as used hereinrefers to a desired compound to which the carbon substrate isbioconverted into. The desired end-product may be the actual compoundsought or an intermediate along another pathway. Exemplary desiredproducts are gluconic acid, 2-keto-D-gluconate; 2,5-diketo-D-gluconate;erythorbic acid; 5-keto-D-gluconate; 2-keto-L-gulonate; tartaric acid;D-ribose; riboflavin; deoxy-ribonucleotides; ribonucleotides; sorbitol;glycerol; sorbose; dihydroxyacetone; aromatic amino acids; aromaticcompounds, such as P-hydroxybenzoic acids, quinones, catechol, indole,indigo, gallic acid, pyrogallol, melanin, adipic acid, andP-aminobenzoic acid; pyridoxine and aspartame. Particularly preferreddesired products are keto derivatives of sorbitol, keto derivatives ofgluconate and keto derivatives of glycerol.

As used herein, the term “bacteria” refers to any group of microscopicorganisms that are prokaryotic, i.e., that lack a membrane-bound nucleusand organelles. All bacteria are surrounded by a lipid membrane thatregulates the flow of materials in and out of the cell. A rigid cellwall completely surrounds the bacterium and lies outside the membrane.There are many different types of bacteria, some of which include, butare not limited to Bacillus, Streptomyces, Pseudomonas, and strainswithin the families of Enterobacteriaceae.

As used herein, the family “Enterobacteriaceae” refers to bacterialstrains having the general characteristics of being gram negative andbeing facultatively anaerobic. For the production of ASA intermediates,preferred Enterobacteriaceae strains are those that are able to produce2,5-diketo-D-gluconic acid from D-glucose or carbon sources which can beconverted to D-glucose by the strain. (Kageyama et al., International J.Sys. Bacteriol. 42:203 (1992)). Included in the family ofEnterobacteriaceae are Erwinia, Enterobacter, Gluconobacter, Klebsiella,Escherichia and Pantoea. In the present invention, a preferredEnterobacteriaceae fermentation strain for the production of ASAintermediates is a Pantoea species. The genus Pantoea includes P.agglomerans, P. dispersa, P. punctata, P. citrea, P. terrea, P. ananasand P. stewartii and in particular, Pantoea citrea. Pantoea citrea canbe obtained from ATCC (Manassas, Va.) for example ATCC No. 39140.Pantoea citrea has sometimes been referred to as Erwinia herbicola orAcetobacter cerenius. Thus, it is intended that the genus Pantoeainclude species that have been reclassified, including but not limitedto Erwinia herbicola or Acetobacter cerenius.

As used herein the family “Bacillus” refers to rod-shaped bacterialstrains having the general characteristics of being gram positive andcapable of producing resistant endospores in the presence of oxygen.Examples of Bacillus include B. subtilis, B. lichenifonnis, B. lentus,B. circulans, B. lautus, B. amyloliquefaciens, B. stearothermophilus, B.alkalophilus, B. coagulans, B. thuringiensis and B. brevis.

As used herein, the term “recombinant” refers to a host cell that has amodification of its genome, e.g., as by the addition of nucleic acid notnaturally occurring in the organism or by a modification of nucleic acidnaturally occurring in the host cell.

The term “heterologous” as used herein refers to nucleic acid or aminoacid sequences not naturally occurring in the host cell. As used herein,the term “endogenous” refers to a nucleic acid or encoded amino acidnaturally occurring in the host.

The terms “isolated” or “purified” as used herein refer to an enzyme,nucleic acid, protein, peptide or co-factor that is removed from atleast one component with which it is naturally associated. In thepresent invention, an isolated nucleic acid can include a vectorcomprising the nucleic acid.

As used herein, the term “vector” refers to a polynucleotide constructdesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, plasmidsand the like.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include a single-,double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid,or a polymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. The following are non-limiting examples ofpolynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA,rRNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs, uracyl, other sugars and linkinggroups such as fluororibose and thioate, and nucleotide branches. Thesequence of nucleotides may be interrupted by non-nucleotide components.

A polynucleotide or polypeptide having a certain percentage (forexample, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 93%, 95%, 97% or 99%) of“sequence identity” to another sequence means that, when aligned, thatpercentage of bases or amino acids are the same in comparing the twosequences. This alignment and the percent homology or sequence identitycan be determined using software programs known in the art, for examplethose described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubelet al., eds., 1987) Supplement 30, section 7.7.18. A preferred alignmentprogram is ALIGN Plus (Scientific and Educational Software,Pennsylvania), preferably using default parameters, which are asfollows: mismatch=2; open gap=0; extend gap=2. Another sequence softwareprogram that could be used is the TFastA Data Searching Programavailable in the Sequence Analysis Software Package Version 6.0 (GeneticComputer Group, University of Wisconsin, Madison, Wis.).

It is well understood in the art that the acidic derivatives ofsaccharides, may exist in a variety of ionization states depending upontheir surrounding media, if in solution, or out of solution from whichthey are prepared if in solid form. The use of a term, such as, forexample, idonic acid, to designate such molecules is intended to includeall ionization states of the organic molecule referred to. Thus, forexample, “idonic acid”, its cyclized form “idonolactone”, and “idonate”refer to the same organic moiety, and are not intended to specifyparticular ionization states or chemical forms.

The term “culturing” as used herein refers to fermentative bioconversionof a carbon substrate to the desired end-product within a reactorvessel. Bioconversion means contacting a microorganism with a carbonsubstrate to convert the carbon substrate to the desired end-product.

As used herein “ATCC” refers to American Type Culture Collection locatedin Manassas, Va. 20108 (ATCC, www/atcc.org).

As used herein, the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

“A,” “an” and “the” include plural references unless the context clearlydictates otherwise.

PREFERRED EMBODIMENTS

A. Genes, Proteins and Vectors:

The present application concerns isolated nucleic acid moleculesencoding gluconate transporter proteins. In one embodiment, an isolatedpolynucleotide which encodes a gluconate transporter has the nucleicacid sequence set forth in SEQ ID NO:1 or a nucleic acid sequence havingat least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%,95%, 97%, 98% and 99% sequence identity to the sequence of SEQ ID NO:1.Preferably the isolated polynucleotide will have at least 40% sequenceidentity, at least 80% sequence identity or at least 95% sequenceidentity to the nucleic acid sequence set forth in SEQ ID NO:1.Preferably the isolated polynucleotide is obtained from a Pantoea hostcell.

In another embodiment, the isolated polynucleotide which encodes agluconate transporter has the nucleic acid sequence set forth in SEQ IDNO:3 or a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98% and 99% sequenceidentity to the sequence of SEQ ID NO:3. Preferably, the isolatedpolynucleotide will have at least 40% sequence identity, at least 80%sequence identity, or at least 95% sequence identity to the nucleic acidsequence, set forth in SEQ ID NO:3. Preferably, the isolatedpolynucleotide is obtained from a Pantoea host cell.

An isolated polynucleotide according to the invention will encode agluconate transporter protein having the amino acid sequence set forthin SEQ ID NO:2 or a gluconate transporter protein having at least 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98% and99% sequence identity to the sequence of SEQ ID NO:2. Preferably theisolated polynucleotide encodes a gluconate transporter protein havingthe sequence as set forth in SEQ ID NO:2 or encodes a gluconatetransporter protein having at least 40% sequence identity, at least 80%sequence identity or at least 95% sequence identity to SEQ ID NO:2. Thegluconate transporter protein having the amino acid sequence as setforth in SEQ ID NO:2 is designated herein as GntU.

An isolated polynucleotide according to the invention may also encode agluconate transporter protein having the amino acid sequence set forthin SEQ ID NO:4 or a gluconate transporter protein having 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98% and 99%sequence identity to the sequence of SEQ ID NO:4. Preferably, theisolated polynucleotide encodes a gluconate transporter protein havingthe sequence as set forth in SEQ ID NO:4 or encodes a transporterprotein having at least 80% sequence identity or at least 95% sequenceidentity to SEQ ID NO:4. The protein having the amino acid sequence asset forth in SEQ ID NO:4 is designated herein as IdnT. Additionally, inone embodiment this gluconate transporter and those having at least 80%sequence identity thereto are also able to facilitate the transport ofidonate across the inner cellular membrane of a host bacterial cell.

One of skill in the art is well aware of the degeneracy of the geneticcode and that an amino acid may be coded for by more than one codon.These variations are include as part of the invention herein.

A further embodiment of the invention includes an isolated gluconatetransporter protein comprising the amino acid sequence set forth in SEQID NO:2 or an amino acid sequence having at least 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 0.80%, 85%, 90%, 93%, 95%, 97%, 98% and 99% sequenceidentity to the sequence of SEQ ID NO:2. In one embodiment, thegluconate transporter will have the amino acid sequence set forth in SEQID NO:2 or at least 40%, at least 80% or at least 95% sequence identitythereof. The gluconate transporter protein set forth in SEQ ID NO:2 wasobtained from Pantoea. In another embodiment, the invention includes anisolated gluconate transporter protein comprising the amino acidsequence set forth in SEQ ID NO:4 or an amino acid sequence having atleast 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%,97%, 98% and 99% sequence identity to the sequence of SEQ ID NO:4. Inone embodiment, the gluconate transporter will have the amino acidsequence set forth in SEQ ID NO:4 or at least 80%.sequence thereto. Inthis embodiment the gluconate transporter will also facilitate thetransport of idonate. The gluconate transporter protein set forth in SEQID NO:4 was obtained from Pantoea. In one embodiment, a preferredgluconate transporter is a transporter obtained from bacterial strainsof Pantoea, particularly a strain of P. citrea or P. agglomerans.

A gluconate transporter according to the invention will catalyze thetransport of gluconate but also may catalyze the transport of thesugar-keto acids, KDG, KLG, 5-KDG, D-Tagaturonic acid, and D-Fructuronicacid. Further a gluconate transporter according to the invention mayalso catalyze the transport of other sugar acids, such as idonic acid,galacturonic acid, gulonic acid and/or glucuronic acid.

Gluconate transporters according to the invention are members of theGntP family of transporters. Over 100 gluconate transporter proteinsfrom various microorganisms have been identified. These microorganismsinclude Escherichia, Bacillus, Haemophilus, Pseudimonas and Clostridium.(Reference is made to Yamada et al., (1996) Biosci. Biotech. Biochem.60:1548-1550; Bausch et al., (1998) J. Bacteriol. 180:3701-3710; Reizeret al., (1991) Mol. Microbiol. 5:1081-1089 and Peekhaus et al., (1997)FEMS Microbiol. Lett. 147:233-238). The overall identity of some of thedisclosed gluconate transporter proteins with the amino acid sequenceillustrated in SEQ ID NO:2 is less than about 34% identity and for SEQID NO:4 less than about 76.3% identity (FIG. 10).

Naturally occurring gluconate transporter proteins have about 12 to 14transmembrane domains and typically are about 400 to 450 amino acids inlength. In this embodiment, a preferred isolated gluconate transporteris a protein obtained from bacterial strains of Pantoea, particularly astrain of P. citrea or P. agglomerans.

Methods useful for identifying gluconate transporter proteins found inbacterial microorganisms such as Pantoea species are well known in theart and would include hybridization studies. Thus, for example, nucleicacid sequences which hybridize under high stringency conditions to agluconate transporter gene identified in FIGS. 4 and 6 or a complementthereof, may also encode proteins that function as a gluconatetransporter. Hybridization includes the process by which a strand ofnucleic acid joins with a complementary strand through base pairing.High stringency conditions are known in the art and see for example,Maniatis, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d Edition(1989) and SHORT PROTOCOLS IN MOLECULAR BIOLOGY, ed Ausubel et al.Stringent conditions are sequence dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY—HYBRIDIZATION WITH NUCLEIC ACID PROBES, Overview of Principlesof Hybridization and the Strategy of Nucleic Acid Assays (1993).Generally stringent conditions are selected to be about 5 to 10 degreeslower than the thermal melting point Tm for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH and nucleic acid concentration) at which 50% of theprobes complementary to the target, hybridize to the target sequence atequilibrium. Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least 30° C. for short probes (e.g. for 10 to 50nucleotides) and at least about 60° C. for long probes (e.g. greaterthan 50 nucleotide). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In anotherembodiment, less stringent hybridization conditions are used. Forexample moderate or low stringent conditions may be used.

PCR may also be used to screen for homologous sequences of gluconatetransporters and reference is made to Chen et al., (1995) Biotechniques18(4):609-612. Exemplary probe oligonucleotides that may be used fordetecting gluconate transporter molecules include the polynucleotidesset forth in SEQ ID NO:1 or SEQ ID NO:3 or fragments thereof. Thesefragments may include at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 65,70, 75, 80, 85, 90 or more contiguous nucleotides from the referencesequence of SEQ ID NO:1 or SEQ ID NO:3. Additionally, an oligonucleotideuseful in the present invention may comprise a nucleotide sequenceencoding a polypeptide having at least 5, 10, 15, 20, 25, 30 or morecontiguous amino acid residues of either SEQ ID NO:2 or SEQ ID NO:4.Other methods include protein bioassay or immunoassay techniques whichinclude membrane-based, solution-based, or chip-based technologies forthe detection and/or quantification of the nucleic acid or protein.

Gluconate transporter activity can be determined by a variety ofmethods. For example, using gluconate uptake assays and detection ofmetabolic products of gluconate or idonate such as by using HPLC asdescribed further in the examples.

Moreover, it is believed that within the cells of the same species orstrain, more than one gluconate transporter protein may be involved inthe transport of gluconate into the cytoplasm. In one example, a familyof gluconate transporter proteins of which GntU and IdnT are memberswould comprise a gluconate transporter system in a Pantoea cell.

Transporter proteins function by moving substrates across a cellularmembrane. As shown in FIG. 2, the function of a gluconate transporter isto facilitate transport of a substrate, such as gluconate into thecytoplasm where it is then made available for cellular metabolism. Byreducing the transport of gluconate into the cytoplasm by rendering agluconate transporter gene non-functional, more substrate will beavailable for oxidative production of keto-sugars and ASA intermediatesand particularly for KLG bioproduction.

B. Bacterial Host Cells:

Particularly preferred bacterial host cells according to the inventionare Enterobacteriaceae cells and particularly E. coli and Pantoea cells.Particularly preferred Pantoea cells are P. citrea and P. aggolmerans(U.S. Pat. No. 5,032,514). Bacillus sp. may also serve as host cells.

In one embodiment, an altered bacterial cell of the invention comprisesat least one non-functional endogenous gluconate transporter. In oneexample, the altered bacterial cell would include a non-functionalgluconate transporter wherein the gluconate transporter in thecorresponding unaltered bacterial cell was encoded by a polynucleotideencoding the amino acid sequence set forth in SEQ ID NO:2 or apolynucleotide encoding a gluconate transporter having an amino acidsequence of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98% or 99% identity with the sequence of SEQ ID NO:2. Ina further embodiment, the gluconate transporter in the unalteredbacterial host cell will be encode by a polynucleotide having thenucleic acid sequence of at least 80%, 85%, 90%, 95%, 97%, 98% and 99%identity with the sequence of SEQ ID NO:1.

In another example, the altered bacterial cell would include anon-functional gluconate transporter wherein the gluconate transporterin the corresponding unaltered bacterial cell was encoded by apolynucleotide encoding the amino acid sequence set forth in SEQ ID NO:4or a polynucleotide encoding a gluconate transporter having an aminoacid sequence with at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identitywith the sequence of SEQ ID NO:4. In a further embodiment, the gluconatetransporter in the unaltered bacterial host cell will be encoded by apolynucleotide having the nucleic acid sequence of at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% and 99% identitywith the sequence of SEQ ID NO:3.

Altered bacterial cells according to the invention may include more thanone non-functional gluconate transporter protein. Altered cellsaccording to the invention may include two, three, four or morenon-functional gluconate transporter proteins. In some embodiments, atleast two gluconate transporter proteins are rendered non-functional. Inone specific example, a gluconate transporter having at least 90%sequence identity with the sequence SEQ ID NO:2 and a gluconatetransporter having at least 90% sequence identity with the sequence ofSEQ ID NO:4 are rendered non-functional in a bacterial host cell. In asecond specific example, a gluconate transporter having at least 95%sequence identity with the sequence SEQ ID NO:2 and a gluconatetransporter having at least 95% sequence identity with the sequence ofSEQ ID NO:4 are rendered non-functional in a bacterial host cell

If a gluconate transporter is rendered non-functional, the transport ofthe substrate gluconate across the inner cell membrane will besubstantially reduced and in some cases gluconate will not betransported across the inner cell membrane. The reduction of gluconatetransport results in increased availability of the gluconate substratein the oxidative pathway for use in production of ASA intermediates.

C. Methods for Rendering Gluconate Transporters Non-Functional:

In general, methods of rendering chromosomal genes non-functional arewell known and a number of these techniques may be used to inactivate agluconate transporter molecule according to the invention. In an alteredbacterial strain according to the invention, the inactivation of thegluconate transporter genes will preferably be a stable andnon-reverting inactivation. Some of these methods include theintegration of DNA into a host cell and reference is made to Balbas etal., (1996), Gene, 172:65-69 and LeBorge et al., (1998), Gene,223:213-219.

One preferred method of inactivation is a deletion of the gene encodingthe gluconate transporter. The deletion may be partial as long as thesequences left in the chromosome are too short for biological activityof the gene. One method of deleting a gluconate transporter geneaccording to the invention includes constructing a vector which includeshomologous flanking regions of the transporter coding sequence ofinterest. The flanking regions may include from about 1 bp to about 500bp at the 5′ and 3′ ends. The flanking region of the vector may belarger than 500 bp but will preferably not include other genes in theregion of the gene to be deleted which would result in these genes beinginactivated or deleted. The vector construct may be introduced into ahost cell by, for example, transformation and then integrated into thehost cell chromosome. The end result is that the introduced DNA causesthe endogenous gluconate gene to be deleted.

In another embodiment, inactivation is by insertion. Insertionalinactivation includes interruption of the chromosomal coding region. Forexample when gntU is the gene to be inactivated, a DNA construct willcomprise a nucleic acid sequence having the gntU gene interrupted by aselective marker. The selective marker will be flanked on each side bysections of the gntU coding sequence. The DNA construct aligns withessentially identical sequences of the gntU gene in the host chromosomeand in a double crossover event the gntU gene is inactivated by theinsertion of the selective marker. A selectable marker refers to a genecapable of expression in the host microorganism which allows for ease ofselection of those hosts containing the vector. Examples of suchselectable markers include but are not limited to antibiotic resistantgenes such as, erythromycin, kanamycin, chloramphenicol andtetracycline.

In another embodiment, inactivation is by insertion in a singlecrossover event with a plasmid as the vector. For example, a gluconatetransporter chromosomal gene such as gntU is aligned with a plasmidcomprising the gntU gene or part of the gene coding sequence and aselective marker. The selective marker may be located within the genecoding sequence or on a part of the plasmid separate from the gene. Thevector may be integrated into the bacterial chromosome, and the gene isinactivated by the insertion of the vector in the coding sequence.

Inactivation may also occur by a mutation of the gene. Methods ofmutating genes are well known in the art and include but are not limitedto chemical mutagenesis, site-directed mutation, generation of randommutations, and gapped-duplex approaches. (U.S. Pat. No. 4,760,025;Moring et al., Biotech. 2:646 (1984); and Kramer et al., Nucleic AcidsRes. 12:9441 (1984)). Chemical mutagenesis may include the use chemicalsthat affect nonreplicating DNA, such as HNO₂ and NH₂OH, as well asagents that affect replicating DNA, such as acridine dyes. Specificmethods for creating mutants using radiation and chemical agents arewell documented in the art. see, for example T. D. Brock inBIOTECHNOLOGY: A TEXTBOOK OF INDUSTRIAL MICROBIOLOGY, 2nd Ed. (1989)Sinauer Associates, Inc. Sunderland, Mass. After mutagenesis hasoccurred, mutants having the desired property may be selected by avariety of methods, such as random screening or selective isolation on aselective media.

In some embodiments, an expression vector is constructed which containsa multiple cloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. In another preferredembodiment, a gluconate transporter gene is deleted by homologousrecombination.

For example, as shown in FIG. 7, an inactivation cassette is constructedby first cloning a DNA fragment containing the gluconate transportergene, gntU into a vector. To inactivate the gntU gene, an antibioticresistance gene (i.e. a chloramphenicol, Cm^(R) gene) is cloned into aunique restriction site found in the gluconate transporter gene. Theinsertion of the antibiotic marker into the gntU gene interrupts itsnormal coding sequence. The inactivation cassette is transferred to thehost cell chromosome by homologous recombination using a non-replicationR6K vector like pGP704 (Miller et al. (1988) J. Bacteriol.170:2575-2583). The transfer of the cassette into the host cellchromosome is selected by the inclusion of CmR by the host cell. Oncethe inactivation of the gntU gene has been corroborated, the Cm^(R) isremoved from the gntU coding region leaving an interrupting spacer(which in this example includes a copy of a loxP site) in the codingregion, inactivating the coding region. (Palmeros et al., (2000) Gene247:255-264). A similar process is seen in FIG. 8 for inactivation ofthe endogenous idnT gene. However in this example two fragments, oneincluding a loxP-Cm^(R)-idnT cassette and one including a idnD cassetteare ligated together and transformed into the host resulting in both theinterruption of the gluconate transporter, idnT and the gene idnD whichencodes idonate dehydrogenase.

Plasmids which can be used as vectors in bacterial organisms are wellknown and reference is made to Maniatis, et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2d Edition (1989) AND MOLECULAR CLONING: A LABORATORYMANUAL, second edition (Sambrook et al., 1989) and Bron, S, Chapter 3,Plasmids, in MOLECULAR BIOLOGY METHODS FOR BACILLUS, Ed. Harwood andCutting, (1990) John Wiley & Sons Ltd.

A preferred plasmid for the recombinant introduction of polynucleotidesencoding non-naturally occurring proteins or enzymes into a strain ofEnterobacteriaceae is RSF1010, a mobilizable, but not self transmissibleplasmid which has the capability to replicate in a broad range ofbacterial hosts, including Gram− and Gram+bacteria. (Frey et al., 1989,The Molecular Biology of IncQ Plasmids. In: Thomas (Ed.), PROMISCUOUSPLASMIDS OF GRAM NEGATIVE BACTERIA. Academic Press, London, pp. 79-94).Frey et al. report on three regions found to affect the mobilizationproperties of RSF1010 (Frey et al. (1992) Gene 113:101-106).

In a further embodiment, the expression product of an inactivated ordeleted gene may be a truncated protein as long as the protein has achange in its biological activity. The change in biological activitycould be an altered activity, but preferably is loss of biologicalactivity.

D. Additional Gene Modifications:

In one embodiment, altered bacterial cells according to the inventionmay include further modifications of chromosomal genes. Thesemodifications to a bacterial host cell may have been realized prior to,simultaneously with, or after inactivation of one or more gluconatetransporter genes. The chromosomal modifications may includeinactivations, such as deletions or interruptions of endogenouschromosomal genes, modifications resulting in increased expression ofendogenous chromosomal genes, and inclusion of heterologous genes. Morespecific examples of modifications to altered host cells of theinvention include but are not limited to:

-   -   (i) modifications concerning additional gluconate transporter        genes;    -   (ii) modifications to genes encoding glucose transporters;    -   (iii) modifications to genes encoding KDG transporters;    -   (iv) modifications to genes encoding DKG transporters (DKG        permease genes), such as prmA, prmB, PE6, PE1 and YiaX2 (See WO        02/12468; WO 02/12481 and WO 02/12528);    -   (v) gene modifications resulting in overexpression of certain        genes, for example overexpression of DKG permease genes or aroZ        genes, which encode dehydratases; and    -   (vi) modifications to a polynucleotide that uncouples the        catabolic pathway from the oxidative pathway such as by        inactivating an enzyme that phosphorylates D-glucose or        D-gluconate at its 6th carbon and more specifically inactivating        a hexokinase gene, a glucokinase gene or a gluconokinase gene        i.e. gntK and/or glkA and reference is made to WO 02/081440.

In certain preferred embodiments, an altered bacterial strain accordingto the invention will include an inactivated gntK and/or an inactivatedg/kA gene. In another preferred embodiment, an altered bacterial strainaccording to the invention will include an overexpressed DKG permease.

In certain embodiments, the altered bacterial strain may embody twoinactivated genes, three inactivated genes, four inactivated genes, fiveinactivated genes, six inactivated genes or more. The inactivated genesmay be contiguous to one another or may be located in separate regionsof the bacterial chromosome. An inactivated chromosomal gene may have anecessary function under certain conditions, but the gene is notnecessary for bacterial strain viability under laboratory conditions.Preferred laboratory conditions include but are not limited toconditions such as growth in a fermentator, in a shake flask, in platemedia or the like.

In some embodiments, the altered bacterial host cells are recombinantPantoea cells which comprise a first inactivated endogenous gluconatetransporter gene, which prior to inactivation encoded a gluconatetransporter having at least 80%, 90%, and 95% sequence identity with SEQID NO:2, a second inactivated endogenous gluconate transporter gene,which prior to inactivation encoded a gluconate transporter having atleast 80%, 90% and 95% sequence identity with SEQ ID NO:4, and aninactivated endogenous glucokinase gene. In some embodiments, theinactivated gluconate transporter genes will have been deleted from thealtered host cell.

In another embodiment, the host cell may be genetically engineered toinclude genes encoding enzymes known to effect the conversion of glucoseor other ordinary metabolites to 2,5-DKG or 2-KLG from the organismsknown to contain them. Examples of the enzymes effecting the conversionof an ordinary metabolite to 2,5-DKG or 2-KLG are D-glucosedehydrogenase (Adachi, O. et al., (1980) Agric. Biol. Chem., 44:301-308;Ameyama, M. et al., (1981) Agric. Biol. Chem. 45:851-861; Smith et al.(1989) Biochem. J. 261:973; and Neijssel et al., (1989) Antonie VanLeauvenhoek 56(1):51-61); D-gluconate dehydrogenase (McIntire, W. etal., (1985) Biochem. J., 231:651-654; Shinagawa, E. et al., (1976)Agric. Biol. Chem. 40:475-483; Shinagawa, E. et al., (1978) Agric. Biol.Chem. 42:1055-1057; and Matsushita et al. (1979), J. Biochem. 85:1173);5-keto-D-gluconate dehydrogenase and 2-keto-D-gluconate dehydrogenase(Shinagawa, E. et al., (1981) Agric. Biol. Chem., 45:1079-1085 andStroshane (1977) Biotechnol. BioEng. 19(4) 459); and2,5-diketo-D-gluconic acid reductase (U.S. Pat. Nos. 5,795,761;5,376,544; 5,583,025; 4,757,012; 4,758,514; 5,008,193; 5,004,690; and5,032,514).

A preferred altered bacterial strain according to the invention will bean E. coli, Bacillus or Pantoea strain and particularly a Pantoea strainsuch as a P. citrea. Preferably, the altered bacterial strain willcomprise an inactivated endogenous gluconate transporter gene and one ormore of the following: a) an inactivated endogenous glucokinase gene; b)an inactivated endogenous gluconokinase gene; c) an inactivatedendogenous glycerol kinase gene; d) an over-expressed DKG transportergene and e) an inactivated or disrupted glucose transport system, suchas the phosphoenolpyruvate glucose phosphotransferase system (PTS).

In one embodiment when KLG is the desired intermediate, nucleic acidencoding 2,5 DKG reductase (DKGR) is recombinantly introduced into thebacterial fermentation strain. Many species have been found to containDKGR particularly members of the Coryneform group, including the generaCorynebacterium, Brevibacterium, and Arthrobacter. In a furtherembodiment DKGR from Corynebacterium sp. strain SHS752001 (Grindley etal., 1988, Applied and Environmental Microbiology 54: 1770-1775) isrecombinantly introduced into a host strain, such as a Pantoea hoststrain. Production of recombinant 2,5 DKG reductase by Erwinia herbicolais disclosed in U.S. Pat. No. 5,008,193.

Gene transfer techniques for bacterial cells are well known and thesetechniques include transformation, transduction, conjugation andprotoplast fusion. Gene transfer is the process of transferring a geneor polynucleotide to a cell or cells wherein exogenously added DNA istaken up by a bacterium. General transformation procedures are taught inCURRENT PROTOCOLS IN MOLECULAR BIOLOGY (vol. 1, edited by Ausubel etal., John Wiley & Sons, Inc. 1987, Chapter 9) and include calciumphosphate methods, transformation using DEAE-Dextran andelectroporation. A variety of transformation procedures are known bythose of skill in the art for introducing nucleic acids in a given hostcell. (Reference is also made to U.S. Pat. No. 5,032,514; Potter H.(1988) Anal. Biochem 174:361-373; Sambrook, J. et al., MOLECULARCLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press(1989); and Ferrari et al., Genetics, pgs 57-72 in BACILLUS, Harwood etal., Eds. Plenum Publishing Corp).

Transformation of a host cell can be detected by the presence/absence ofmarker gene expression which can suggest whether the nucleic acid ofinterest is present. However, its expression should be confirmed. Forexample, if the nucleic acid encoding a gluconate transporter isinserted within a marker gene sequence, recombinant cells containing theinsert can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with nucleic acidencoding the transporter under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the protein or enzyme as well. Once abacterial microorganism capable of carrying out the conversion asdescribed above has been created, the process of the invention may becarried out in a variety of ways depending on the nature of theconstruction of the expression vectors for the recombinant proteins andupon the growth characteristics of the host.

E. Cell Cultures and Fermentations.

Methods suitable for the maintenance and growth of bacterial cells iswell known and reference is made to the MANUAL OF METHODS OF GENERALBACTERIOLOGY, Eds. P. Gerhardt et al., American Society forMicrobiology, Washington D.C. (1981) and T. D. Brock in BIOTECHNOLOGY: ATEXTBOOK OF INDUSTRIAL MICROBIOLOGY, 2nd ed. (1989) Sinauer Associates,Sunderland, Mass.

Cell Precultures—Typically cell cultures are grown at 25 to 32° C., andpreferably about 28 or 29° C. in appropriate media. Exemplary growthmedia useful in the present invention are common commercially preparedmedia such as but not limited to Luria Bertani (LB) broth, SabouraudDextrose (SD) broth or Yeast medium (YM) broth. These may be obtainedfrom for example, GIBCO/BRL (Gaithersburg, Md.). Other defined orsynthetic growth media may be used and the appropriate medium for growthof the particular bacterial microorganism will be known by one skilledin the art of microbiology or fermentation science. Suitable pH rangespreferred for the fermentation are between pH 5 to pH 8. Preferredranges for seed flasks are pH 7 to pH 7.5 and preferred ranges for thereactor vessels are pH 5 to pH 6. It will be appreciated by one of skillin the art of fermentation microbiology that a number of factorsaffecting the fermentation processes may have to be optimized andcontrolled in order to maximize the ascorbic acid intermediateproduction. Many of these factors such as pH, carbon sourceconcentration, and dissolved oxygen levels may affect enzymaticprocesses depending on the cell types used for ascorbic acidintermediate production.

The production of ASA intermediates can proceed in a fermentativeenvironment, that is, in an in vivo environment, or a non-fermentativeenvironment, that is, in an in vitro environment; or combined in vivo/invitro environments. The fermentation or bioreactor may be performed in abatch process, a Fed-batch process or in a continuous process.

In Vivo Biocatalytic Environment

Biocatalysis begins with culturing an altered host cell according to theinvention in an environment with a suitable carbon source ordinarilyused by Enterobacteriaceae or other bacterial strains. Suitable carbonsources include 6 carbon sugars, for example, glucose, or a 6 carbonsugar acid, or combinations of 6 carbon sugars and/or 6 carbon sugaracids. Other carbon sources include, but are not limited to galactose,lactose, fructose, or the enzymatic derivatives of such.

In addition, fermentation media must contain suitable carbon substrateswhich will include but are not limited to monosaccharides such asglucose, oligosaccharides such as lactose or sucrose, polysaccharidessuch as starch or cellulose and unpurified mixtures from a renewablefeedstocks such as cheese whey permeate, cornsteep liquor, sugar beetmolasses, and barley malt. Additionally, the carbon substrate may beone-carbon substrates such as carbon. While it is contemplated that thesource of carbon utilized in the present invention may encompass a widevariety of carbon containing substrates and will only be limited by thechoice of organism, the preferred carbon substrates include glucose,fructose and sucrose and mixtures thereof. By using mixtures of glucoseand fructose in combination with the altered bacterial strains describedherein, uncoupling of the oxidative pathways from the catabolic pathwaysallows the use of glucose for improved yield and conversion to thedesired ASA intermediate while utilizing the fructose to satisfy themetabolic requirements of the host cells.

In one embodiment, the concentration of the carbon substrate in the seedsolution is from about 55% to about 75% on a weight/weight basis.Preferably, the concentration is from about 60 to about 70% on aweight/weight basis. Most preferably the concentration used is 60% to67% glucose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, vitamins, cofactors and bufferssuitable for the growth or the cultures and promotion of the enzymaticpathway necessary for ascorbic acid intermediate production.

Batch, Fed-Batch and Continuous Fermentations:

The present invention may employ a batch fermentation process, amodified batch fermentation process called Fed-batch or a continuousfermentation process.

A classical batch fermentation is a closed system where the compositionof the media is set at the beginning of the fermentation and not subjectto artificial alterations during the fermentation. At the beginning ofthe fermentation the media is inoculated with the desired bacterialorganism or organisms and fermentation is permitted to occur addingnothing to the system. Typically, however, a “batch” fermentation isbatch with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures, cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in log phase generally are responsible for thebulk of production of desired end products or intermediates.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in T. D. Brock in BIOTECHNOLOGY: ATEXTBOOK OF INDUSTRIAL MICROBIOLOGY, Second Edition (1989) SinauerAssociates, Inc. Sunderland, Mass.

Continuous fermentation is an open system where a defined fermentationmedia is added continuously to a bioreactor and simultaneously an equalamount of conditioned media is removed for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth; Continuous fermentationallows for the modulation of one factor or any number of factors thataffect cell growth or end product concentration. For example, one methodwill maintain a limiting nutrient such as the carbon source or nitrogenlevel at a fixed rate and allow all other parameters to moderate. Inother systems a number of factors affecting growth can be alteredcontinuously while the cell concentration, measured by media turbidity,is kept constant. Continuous systems strive to maintain steady stategrowth conditions and thus the cell loss due to media being drawn offmust be balanced against the cell growth rate in the fermentation.Methods of modulating nutrients and growth factors for continuousfermentation processes as well as techniques for maximizing the rate ofproduct formation are well known in the art of industrial microbiologyand a variety of methods are detailed by Brock, supra.

In one illustrative in vivo example of a pathway in Pantoea, D-glucoseundergoes a series of membrane oxidative steps through enzymaticconversions, which may include the enzymes D-glucose dehydrogenase,D-gluconate dehydrogenase and 2-keto-D-gluconate dehydrogenase to giveintermediates which may include, but are not limited to GA, KDG, andDKG, see FIGS. 1 and 2. These intermediates undergo a series ofintracellular reducing steps through enzymatic conversions, which mayinclude the enzymes 2,5-diketo-D-gluconate reductase (DKGR), 2-ketoreductase (2-KR) and 5-keto reductase (5-KR) to give end products whichinclude but are not limited to KLG and IA. In a preferred embodiment ofthe in vivo environment for the production of ASA intermediates,gluconate is not transported across the host cell membrane which resultsin an increased availability of gluconate for oxidative pathwayproduction of ASA intermediates.

F. Recovery, Identification and Purification of ASA Intermediates:

Methods for detection of ASA intermediates, ASA and ASA stereoisomerssuch as D-araboascorbic acid (erythorbic acid), L-araboascorbic acid,and D-xyloascorbic acid include the use of redox-titration with 2,6dichloroindophenol (Burton et al. (1979) J. Assoc. Pub. Analysts 17:105)or other suitable reagents; high-performance liquid chromatography(HPLC) using anion exchange; and electro-redox procedures (Pachia,(1976) Anal. Chem. 48:364). The skilled artisan will be well aware ofcontrols to be applied in utilizing these detection methods.Alternatively, the intermediates can also be formulated directly fromthe fermentation broth or bioreactor and granulated or put in a liquidformulation. KLG produced by a process of the present invention may befurther converted to ascorbic acid and the KDG to erythorbate by meansknown to those of skill in the art, see for example, Reichstein andGrussner, Helv. Chim. Acta., 17, 311-328 (1934).

G. Increased Yield of Desired Products from the ASA Pathway:

The catabolic pathway is uncoupled from the ASA oxidative pathway toincrease the availability of gluconate or idonate for ASA intermediateproduction. As shown in FIGS. 1 and 2 gluconate can be transportedacross the inner cell membrane to the cytoplasm. Gluconate may then bephosphorylated, for example to gluconate—6- phosphate and made availablefor catabolic metabolism in the pentose pathway. Additionally, gluconatemay be enzymatically reduced to 5-KDG or 2-KDG in the cytoplasm.Inactivation or modification of the levels of a gluconate transporteraccording to the invention by inactivation of the nucleic acid encodingthe same, results in an increased amount of gluconate available for theoxidative ASA production pathway resulting in a desired product. Idonatemay also be transported by the gluconate transporter across the innermembrane into the cytoplasm and through a series of enzymatic steps beconverted to gluconate. This gluconate may then be phosphorylated asdescribed above. Inactivation or modification of the levels of agluconate transporter according to the invention by inactivation of thenucleic acid encoding the same, results in an increased amount ofidonate available for conversion to 2-KLG. (FIGS. 1 and 2).

In an embodiment of the invention, the catabolic pathway is uncoupledfrom the ASA oxidative pathway to increase the production of KDG.Inactivation of the gluconate transporter diminishes the transport ofgluconate across the inner cell membrane and reduces the availability ofgluconate for phosphorylation by a kinase. Inactivation-of the gluconatetransporter gene or modification of the levels of the gluconatetransporter results in the increased yield of a desired product, e.g.KDG.

In another embodiment, the catabolic pathway is uncoupled from theproductive oxidative pathway to increase the production of DKG.Inactivation or modifying the levels of the gluconate transporter bymodifying the nucleic acid or polypeptide encoding the same, results inthe increased yield of the desired product, e.g. DKG.

In a further embodiment, the catabolic pathway is uncoupled from theproductive oxidative pathway to increase the production of KLG.Inactivation or modifying the levels of the gluconate transporter bymodifying the nucleic acid or polypeptide encoding the same, results inthe increased yield of the desired product, e.g. KLG.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of erythorbic acid.Inactivation or modifying the levels of the gluconate transporter bymodifying the nucleic acid or polypeptide encoding the same, results inthe increased yield of DKG which is an intermediate in the synthesis oferythorbic acid.

General Experimental Methods

Cells: All commercially available cells used in the following exampleswere obtained from the ATCC and are identified in the text by their ATCCnumber. Recombinant P. citrea cells used as a control were derived fromATCC 39140. (Also reference is made to Truesdell et al., (1991) J.Bacteriol. 173:6651-6656).

Materials and Methods suitable for the maintenance and growth ofbacterial cultures were found in MANUAL OF METHODS FOR GENERALBACTERIOLOGY (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow,Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips,eds), pp. 210-213. American Society for Microbiology, Washington, D.C.or in Thomas D. Brock in BIOTECHNOLOGY: A TEXTBOOK OF INDUSTRIALMICROBIOLOGY, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass. All reagents and materials used for the growth ofbacterial cells were obtained from Diffco Laboratories (Detroit, Mich.),Aldrich Chemicals (Milwaukee, Wis.) or Sigma Chemical Company. (St.Louis, Mo.) unless otherwise specified.

Ascorbic acid intermediate analysis: The presence of ascorbic acidintermediates, e.g., 2-KLG, was verified by running a HPLC analysis.Fermentation reactor vessel samples were drawn off the tank and loadedonto Dionex (Sunnyvale, Calif., Product No. 043118) Ion Pac AS 10 column(4 mm times 250 mm) connected to a Waters 2690 Separation Module and aWaters 410 Differential Refractometer (Milford, Mass.).

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto. All references and patent publications referred to herein arehereby incorporated by reference.

EXAMPLES Example 1 Inactivation of the gntU ORF Encoding a GluconateTransporter in P. citrea

Cloning of the gntKU Operon

A 1934-bp DNA fragment containing the gntK and gntU structural genesfrom Pantoea citrea was amplified by PCR with GntKUF1 (SEQ ID NO:5) plusGntKUR1 (SEQ ID NO:6) primer pairs using standard techniques (Sambrooket al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press (1989) 2nd Ed.). In addition to the structural genes,the PCR fragment carries 27 bp upstream of the gntK gene and 23 bpdownstream of the gntU (FIG. 7). This PCR product was cloned into theHindIII site of pCL1920 vector resulting in plasmid pCL-GntKU2 (6489-bp)(Lerner et al., (1990) Nucleic Acids Res. 18:4631). This was constructedinto E. coli TOP10 strain (Invitrogen, Carlsbad, Calif.) and checked byEcoRI and PstI restriction enzymes for correct insert and orientation.

Construction of the Plasmid Used to Inactivate gntU:

In general, the strategy used to inactivate genes by homologousrecombination with a vector has been delineated before and reference ismade to Miller et al., (1988) J. Bacteriol. 170:2575-2583. This generalapproach was used to inactivate gntU.

The pCL-GntKU2 plasmid described above was used for inactivation of thechromosomal gntU. pCL-GntKU2 was digested with NsiI enzyme (RocheApplied Science) to remove the 435-bp region from the middle of the gntUgene. A cat-loxP, 1080-bp cassette (Palmeros et al., (2000) Gene247:255-264) was inserted into the construct resulting in plasmidpCLKUCat1 (7131-bp). The orientation of the cat-loxP cassette waschecked with Scal1+Spel restriction enzymes. The 2580-bp gntKU+cat-loxPregion was amplified by PCR using primers GntKUF1 (SEQ ID NO:5) andGntKUR1 (SEQ ID NO:6), and ligated with the 502-bp DNA fragment thatcontains the minimal R6K origin of replication (ori). This particularDNA was obtained by PCR using plasmid pGP704 (Miller et al., (1988) J.Bacteriol. 170:2575-2583) as PCR substrate with primers

R6K1 (TGTCAGCCGTTAAGTGTTCCTGTG) (SEQ ID NO:18) and R6K2(CAGTTCAACCTGTTGATAGTACG). (SEQ ID NO:19)The ligation product was transformed into E. coli PIR1 strain(Invitrogen, Carlsbad, Calif.) to generate the plasmid pKUCatR6-1(3087-bp). This plasmid was used to inactivate the gntU gene. In thisconstruct, a 650-bp and 850-bp region of homology is available at the5′- and 3′-end of the gntU gene, respectively, for homologousrecombination into the P. citrea chromosome. This recombination eventshould not, affect the proximal gntK gene in the gntKU operon.Transformation into P. citrea Strains:After checking pKUCatR6-1 with Aatll+Accl restriction enzymes, theplasmid was used to transform the strain MDP41 of P. citrea. Alltransformations were done by standard electroporation. (Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor LaboratoryPress (1989) 2nd Ed.). Transformants were selected on LA+Cm12 plates (LAis LB plus agar+plates with 12 ppm chloramphenicol (Cm). Hundreds ofCm-resistant (CmR) colonies appeared. Four colonies were checked by PCRanalysis as described below.Verification of the Altered gntU Strains:To distinguish between single and double crossover events, the junctionpoints were checked with one outside primer plus one cat gene specificprimer. With GntKUF3 (outside) (SEQ ID NO:)+cat4 (SEQ ID NO:11) andGntKUF4 (outside) (SEQ ID NO:8)+cat4 (SEQ ID NO:11) primer pairs, allfour transformants amplified a 0.99-kb and a 0.95-kb fragment,respectively. With cat3 (SEQ ID NO:10)+GntKUR3 (outside) (SEQ ID NO:9),only NO:2 and NO:4 transformants amplified the expected 1.45-kb. Thus,NO:2 and NO:4 transformants had undergone a double cross-over eventwherein the gntU was deleted. In contrast, NO:1 and NO: 3 transformantshad undergone a single cross-over event wherein the gntU remained intactat the gntU locus.

The 1.5-kb and 1.0-kb PCR products obtained with GntKUF4+cat4 (SEQ IDNO:8+SEQ ID NO:11) and cat3+GntKUR3 (SEQ ID NO:10+SEQ ID NO:9) primerpairs were digested with BglII and PstI enzymes, respectively, to verifythe authenticity of the gntU deletion of these strains. Expecteddigestion patterns were obtained with both enzymes.

The NO: 2 transformant was named MDP41U2 and further used inexperiments.

Removal of the Chloramphenicol Marker (CmR) from MDP41U2.

The CmR marker from strain MDP41U2 was removed by bacteriophage P1Crerecombinase using the procedure disclosed in Palmeros et al. (2000) Gene247:255-264. Briefly, plasmid pJW168 expressing the IPTG-inducible Crerecombinase was introduced into MDP41U2 strain by electroporation, Theremoval of CmR marker was achieved by screening colonies forchloramphenicol sensitivity (CmS) phenotype on antibiotic-free LA platesfollowed by PCR verification. Finally plasmid pJW168 was eliminated fromthe CmS strain by growing the strains at non-permissible temperature andchecking for carbenicillin-sensitivity (CarbS) of the strains. Themarker-less gntU deletion strain was named MDP41UF and was used foradding the idnT deletion.

TABLE 1 Primers used for gntU Deletion SEQ ID Name Sequence (5′ to 3′)NO: GntKUF1 TTGCTGCATCAACAGTCGGAG 5 GntKUR1 GTTATTCCGGCACAAGCAGCC 6GntKUF3 GCGCTGGCCACGGTAGTTA 7 GntKUF4 CGAAGAAGCCGCCAGGATGTTA 8 GntKUR3CCGGCGGTATTATTTTGTTTCTGA 9 Cat3 GAAAGTTGGAACCTCTTACGTGCCG 10 Cat4ACAACAGTACTGCGATGAGTGGCAG 11

Example 2 Inactivation of the idnT ORF Encoding a Gluconate Transporterin Pantoea citrea

Construction of the idnDT Deletion Plasmid:

Primers PcldnT-1 (SEQ ID NO:12) and PcldnT-2 (SEQ ID NO:13) weredesigned approximately 1 kb upstream and 1 kb downstream of the idnTgene (SEQ ID NO:3). The amplification of PCR product of 1473-bp wasobtained and cloned into pUniBluntV5-His-Topo (Echo Cloning System fromInvitrogen). The resulting plasmid was transformed into E. coli PIR1strain (Invitrogen, Carlsbad, Calif.) and appropriate restrictionenzymes were used to check for the correct orientation of the gene.

IdnT gene was interrupted using BgII enzyme followed by insertion of acat-loxP (1080-bp) cassette (Palmeros et al., (2000) Gene 247:255-264)resulting in plasmid pEkIdnTCat2 (4813-bp). As described in example 1,the orientation of the cat-loxP cassette was checked with appropriaterestriction enzymes.

The following 2 primers PcIdnD5 (SEQ ID NO:14)+PcIdnD6 (SEQ ID NO:15)were used to amplify the IdnD gene which is upstream of the idnT gene. A1891-bp fragment encompassing the IdnD gene was amplified by PCR usingknown techniques and was cloned into pUniBluntV5-His-Topo (Echo CloningSystems, Invitrogen) resulting in a plasmid called pIdnD56-1.

Construction of pEkIdnD56-1 and pEkIdnTCat2 to Obtain the Final PlasmidpEkIdnDTCat2.

pEkidnTCat2 was digested with BssH II and NsiI enzymes to remove a1883-bp region of R6k ori and partial Km resistance gene, and theremaining 2930-bp of IdnT gene with the cat-loxP cassette was purifiedand ligated to IdnD gene as follows:

pIdnD56-1 was digested with BssH II and NsiI enzymes and a 2576-bpfragment containing the IdnD gene with R6K ori region was purified andligated to 2930-bp of IdnT gene with the cat-loxP cassette followed bytransformation into E. coli PIR1 strain to generate the final plasmidpEkIdnDT Cat2 (5506-bp) that was used for idnDT deletion. PlasmidpEkIdnDT Cat2 was verified with NcoI+Bgl II enzymes.

Transformation of pEkIdnDT Cat2 into P. citrea MDP41UF Strain.

The plasmid pEkIdnDTCat2 was transformed into P. citrea MDP41U2 strainusing the same procedure as described above in example 1 andtransformants were selected on LA+Cm12. Hundreds of CmR colonies wereobtained followed by picking and patching simultaneously onto LA+Cm12and LA+Kan50 plates to screen for kanamycin-sensitive (Kan^(S)) clones.Seven (Cm^(R)Kan^(S)) colonies were checked by PCR for the deletion ofthe idnDT region with appropriate primers as described herein and onecolony was subsequently named MDP41UDT. (FIG. 8)

Verification of the idnDT Deletion in MDP41UDT:

A set of outside primers (PcIdnD7 (SEQ ID NO:17)+PcIdnT4 (SEQ ID NO:16))were used to verify the idnDT deletion. Another set of outside primersplus one cat gene specific primer (pcIdnD7 (SEQ ID NO:19)+Cat4 (SEQ IDNO:11)) & (PcIdnT4 (SEQ ID NO:16)+Cat3 (SEQ ID NO:10)) were used toverify the junctions of the deleted regions with the cat-loxP region.

Plasmid pD92-A (14.2-kb) was derived from plasmid pD92 (12.6-kb). pD92was constructed by cloning the structural gene for the2,5-diketo-D-gluconate reductase B from Corynebacterium sp. (Grindley etal., (1988) Appl. Environ. Microbiol. 54:1770-1775) into pl43 (U.S. Pat.No. 5,008,193). The structural gene for permA gene described in WO02/12468, WO 02/12528 and WO 02/12481 was introduced into the uniqueNotI site of plasmid pD92. This site is located in the structural geneof the streptomycin resistant (Strep^(R)) gene and the cloning of permAcaused its inactivation. In plasmid pD92-A, the expression of permA geneis driven by its native promoter. Plasmid pD92-A was then transformedinto MDP41UDT strain, resulting in MDP41UDT/pD92-A.

TABLE 2 Primers used for IdnT gene deletion Name Sequence 5′ to 3′ SEQID NO: PcldnT-1 TAGGTATAGCCGAAGGGATGACAC SEQ ID NO:12 PcldnT-2AGAGCCTTTGCCTTTGATAACAGC SEQ ID NO:13 PcldnD5 CTTTGGCCGCTGAACTGACGAGATSEQ ID NO:14 PcldnD6 TATAAACAGAAAAGGACAGATGAG SEQ ID NO:15 PcldnT-4TACCAGCCGCATACCGATACACC SEQ ID NO:16 PcldnD7 CCGGTTATTCGCGTTATGTG SEQ IDNO:17 Cat3 GAAAGTTGGAACCTCTTACGTGCCG SEQ ID NO:10 Cat4ACAACAGTACTGCGATGAGTGGCAG SEQ ID NO:11

Example 3 Shake Flask Experiments

Three strains of Pantoea citrea were used in this example:

-   -   (1) Strain 139-2A/pD92 was used as a control.    -   (2) Strain MDP41/pD92A disclosed in WO 02/081440 which includes        an interruption in glkA.    -   (3) Strain MDP41UDT/pD92-A (as described above in examples 1        and 2) which includes an interruption in g/kA and a deletion in        gntU and idnT.

These strains were grown in the following medium (KH₂PO₄(12.0 g/L);K₂HPO₄ (4.0 g/L); MgSO₄.7H₂O (2.0 g/L); Difco Soytone (2.0 g/L); Sodiumcitrate (0.1 g/L); Fructose (5.0 g/L); (NH₄)₂SO₄ (1.0 g/L); Nicotinicacid (0.02 g/L); FeCl₃.6H₂O (5 mL/L of a 0.4 g/L stock solution) andTrace salts (5 mL/L—of the following solution: 0.58 g/L ZnSO₄.7H₂O, 0.34g/L MnSO₄.H₂O, 0.48 g/L Na₂MoO₄.2H₂O) with fructose as the sole carbonsource or with a mixed carbon source (fructose, gluconate and DKG) inthe range from 0.1% to 1.0%. Cells were grown at a temperature between20-37° C., (preferably below 30° C.) and at a pH 7.0 (but a pH range of5.0 to 8.0 should also work).

Gluconate Uptake Biochemical Assay:

Samples of the fermentation broth containing cells of the strainsdesignated above were withdrawn from the respective shake flasks andwere quenched on ice-water bath. The fermentation broth was centrifugedat refrigerating conditions and the supernatant discarded. The cellpellet was washes with 0.95% ice cold saline solution followed by twowashed with gluconate uptake assay buffer (100 mM ice-cold potassiumphosphate, pH 6.9 containing 10 mM fructose). Cells were suspended inthe same assay buffer to an optical density of 12 at 550 nm, and thenwere incubated at room temperature and preferably 28° C. The gluconateuptake assay was started by mixing the cell suspension solution with C¹⁴enriched radio-isotoped gluconate. The time course of gluconate uptakewas performed using vacuum/filter based quenching using ice-coldgluconate uptake assay buffer. Gluconate uptake measurements were doneby radioisotope incorporation in the cells and the data obtained plottedagainst time to give the gluconate uptake rate. (See FIG. 9).

The results support that deletion of gntU and idnT were effective inlowering the gluconate uptake rate. In this experiment, the rate ofgluconate uptake rate for the control (139-2A/pD92) and the strainMDP41/pD92-A were similar. The gluconate uptake rate of MDP41UDT/pD92-Awhich includes the interruption of glkA, deletion of gntU and deletionof idnT further reduced gluconate uptake rate by 70% compared to thecontrol.

Example 4 Fermentor Experiments with Pantoea citrea Host Cells Having aDeletion of gntU and idnT

Seed Train: A vial of culture containing the indicated strains which wasstored in liquid nitrogen, was thawed in air and 0.75 mL was added to asterile 2-L Erlenmeyer flasks containing 500 mL of seed medium. Flaskswere incubated at 29° C. and 250 rpm for 12 hours. Transfer criteria isan OD₅₅₀ greater than 2.5.Seed flask medium—A medium composition was made according to thefollowing: KH₂PO₄ (12.0 g/L); K₂HPO₄ (4.0 g/L); MgSO4.7H₂O (2.0 g/L);Difco Soytone (2.0 g/L); Sodium citrate (0.1 g/L); Fructose (5.0 g/L);(NH₄)₂SO₄ (1.0 g/L); Nicotinic acid (0.02 g/L); FeCl₃.6H₂O (5 mL/L of a0.4 g/L stock solution) and Trace salts (5 mL/L—of the followingsolution: 0.58 g/L ZnSO₄.7H₂O, 0.34 g/L MnSO₄.H₂O, 0.48 g/LNa₂MoO₄.2H₂O).

The pH of the medium solution was adjusted to 7.0±0.1 unit with 20%NaOH. Tetracycline HCl was added to a final concentration of 20 mg/L (2mL/L of a 10 g/L stock solution). The resulting medium solution was thenfilter sterilized with a 0.2, filter unit. The sterile medium was addedto a previously autoclaved flask.

Production Fermentor—Additions to the reactor vessel prior tosterilization included: KH₂PO₄ (3.5 g/L); MgSO₄.7H₂O (1.0 g/L);(NH₄)₂SO₄ (0.92 g/L); Mono-sodium glutamate (15.0 g/L); ZnSO₄7H₂O (5.79mg/L); MnSO₄.H₂O (3.44 mg/L); Na₂MoO₄.2H₂O (4.70 mg/L); FeCl₃.6H₂O (2.20mg/L); Choline chloride (0.112 g/L) and Mazu DF-204 (0.167 g/L) anantifoaming agent.

The above constituted media was sterilized at 121° C. for 45 minutes.After tank sterilization, the following additions were made to thefermentation tank: Nicotinic acid (16.8 mg/L); Ca-pantothenate (3.36mg/L); Glucose (25 g/L) and Fructose (25 g/L).

The final volume after sterilization and addition of post-sterilizationcomponents was 6.0 L. The so prepared tank and medium were inoculatedwith the full entire contents from seed flasks prepared as described togive a volume of 6.5 L.

Growth conditions were at 29° C. and pH 6.0. Agitation rate, backpressure, and air flow are adjusted as needed to keep dissolved oxygenabove zero. When the sugars initially batched into the medium have beenexhausted, a fed-batch process as previously described herein isemployed. In this example both glucose and fructose are used assubstrates in contrast to the standard fed-batch process where onesubstrate is employed.

As observed in Table 3 below, the yield of KLG obtained inMDP41UDT/pD92-A was greater than the yield obtained in the controlMDP41/pD92-A.

TABLE 3 Fermentation Yield Performance Metrics for Yield on KLG onSugars Average Yield Standard Mini- Strain (g/g) Deviation Maximum mumNumber MDP41/pD92-A 0.601 0.021 0.636 0.581 8 MDP41UDT/pD92-A 0.6430.009 0.654 0.630 5 t-test 0.001 MDP41/pD92-A is the control strain andincludes a glkA interruption. Strain MDP41UDT/pD92-A is derived fromMDP41/pD92 and includes a glkA interruption, a gntU deletion and an idnTdeletion.

A further unexpected result included the increased production of KLG inrelation to the formation of IA. KLG was measured as described above inthe general experimental methods. Typically KLG is reduced furtherwithin strains to produce idonic acid. After fermentation is complete,air can be circulated through the fermentor and a membrane bounddehydrogenase oxidizes the IA back to KLG. With the deletion of gntU andidnT, the ratio of KLG to IA is increased thus reducing the timerequired for the back conversion of IA to KLG (Table 4). Reference isalso made to FIG. 2.

TABLE 4 Ratio of KLG to IA KLG IA STRAIN (G/L) (G/L) RATIO MDP41/PD92-A189 75 2.52 MDP41UDT/PD92-A 214 47 4.55

1. A method for enhancing the level of production of an ascorbic acid(ASA) intermediate in a Pantoea bacterial cell comprising: a)recombinantly inactivating a gene encoding a gluconate transporter,wherein said transporter has at least 95% sequence identity with thesequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 4 in saidbacterial cell to obtain a recombinantly altered bacterial cell and; b)culturing the recombinantly altered bacterial cell under suitableculture conditions in the presence of a carbon source to allow theproduction of an ASA intermediate, wherein the level of production ofthe ASA intermediate in the recombinantly altered bacterial cell isenhanced compared to the level of production of said ASA intermediate inthe bacterial cell when grown under essentially the same cultureconditions, wherein the ASA intermediate is selected from the groupconsisting of gluconate, 2-keto-D-gluconate (2-KDG),2,5-diketo-D-gluconate (2,5-DKG), 2-keto-L-gulonic acid (2-KLG),5-keto-D-gluconate (5-KDG), and L-idonic acid (IA).
 2. The methodaccording to claim 1, wherein the ASA intermediate is 2-KDG.
 3. Themethod according to claim 1, wherein the ASA intermediate is 2-KLG. 4.The method according to claim 1, wherein two gluconate transporters areinactivated and wherein a first gluconate transporter gene encodes agluconate transporter having at least 95% sequence identity to SEQ IDNO: 2 and a second gluconate transporter gene encodes a gluconatetransporter having at least 95% sequence identity to SEQ ID NO:
 4. 5.The method according to claim 1, wherein the recombinantly alteredbacterial cell is an Escherichia coli cell.
 6. The method according toclaim 1, wherein the Pantoea cell is a Pantoea citrea cell.
 7. Themethod according to claim 1, wherein the gluconate transporter has atleast 95% sequence identity to SEQ ID NO:
 2. 8. The method according toclaim 1, wherein the gluconate transporter has at least 98% sequenceidentity to SEQ ID NO:
 2. 9. The method according to claim 1, whereinthe gluconate transporter has at least 95% sequence identity to SEQ IDNO:
 4. 10. The method according to claim 1, wherein the gluconatetransporter has at least 98% sequence identity to SEQ ID NO:
 4. 11. Themethod according to claim 1, wherein the gene encodes a gluconatetransporter having the amino acid sequence of SEQ ID NO:
 2. 12. Themethod according to claim 1, wherein the gene encodes a gluconatetransporter having the amino acid sequence of SEQ ID NO:
 4. 13. Themethod according to claim 1, wherein the carbon source is selected fromthe group consisting of glucose, gluconate, fructose, galactose andcombinations thereof.
 14. The method according to claim 1, wherein thegene encoding the glucose transporter has been deleted.
 15. The methodaccording to claim 1, wherein the altered bacterial cell is a straincomprising a recombinantly introduced 2,5-diketo-D-gluconic acidreductase.